MORBIDITY AND MORTALITY OF HAWAIIAN GEESE (BRANTA SANDVICENSIS) AND LAYSAN ALBATROSS (PHOEBASTRIA IMMUTABILIS) ASSOCIATED WITH RETICULOENDOTHELIOSIS VIRUS.

Only one virus, Avipox, has been documented previously in wild birds in Hawaii. Using immunohistochemistry and PCR, we found that two native threatened Hawaiian Geese (Branta sandvicensis), one with multicentric histiocytoma and the other with toxoplasmosis, and one Laysan Albatross (Phoebastria immutabilis) with avian pox were infected with reticuloendotheliosis virus (REV). The virus was isolated from one of the geese by cell culture. Surveys of other Hawaiian geese with various pathologies, avian pox cases, and pox viral isolates using PCR failed to reveal REV, suggesting that the virus is uncommon, at least in samples examined. The full genome of the Gag, Pol, and Env genes were sequenced for all three infected birds and revealed geographic divergence of the Pol gene, suggesting it to be under strong selective pressure. Our finding of REV in Hawaii makes this only the second virus documented in native Hawaiian birds associated with pathology. Moreover, the presence of REV in a pelagic seabird is unusual. Future surveys should seek the reservoir of the virus in efforts to trace its origins.

Cytology reveals diverse cell morphotypes and cellin-cell interactions in normal collector sea urchins Tripneustes gratilla.

Echinoderms such as sea urchins are important in marine ecosystems, particularly as grazers, and unhealthy sea urchins can have important ecological implications. For instance, unexplained mortalities of Diadema antillarum in the Caribbean were followed by algal overgrowth and subsequent collapse of coral reef ecosystems. Unfortunately, few tools exist to evaluate echinoderm health, making management of mortalities or other health issues problematic. Hematology is often used to assess health in many animal groups, including invertebrates, but is seldom applied to echinoderms. We used a standard gravitometric technique to concentrate fixed coelomocytes from the collector sea urchin Tripneustes gratilla onto microscope slides, permitting staining and enumeration. Using Romanowsky stain and electron microscopy to visualize cell details, we found that urchin cells could be partitioned into different morphotypes. Specifically, we enumerated phagocytes, phagocytes with perinuclear cytoplasmic dots, vibratile cells, colorless spherule cells, red spherule cells, and red spherule cells with pink granules. We also saw cell-in-cell interactions characterized by phagocytes apparently phagocytizing mainly the motile cells including red spherule cells, colorless spherule cells, and vibratile cells disproportionate to underlying populations of circulating cells. Cell-in-cell interactions were seen in 71% of sea urchins, but comprised <1% of circulating cells. Finally, about 40% of sea urchins had circulating phagocytes that were apparently phagocytizing spicules. The coelomic fluid collection and slide preparation methods described here are simple, field portable, and might be a useful complementary tool for assessing health of other marine invertebrates, revealing heretofore unknown physiological phenomena in this animal group.

A novel host-adapted strain of Salmonella Typhimurium causes renal disease in olive ridley turtles (Lepidochelys olivacea) in the Pacific.

Salmonella spp. are frequently shed by wildlife including turtles, but S. enterica subsp. enterica serovar Typhimurium or lesions associated with Salmonella are rare in turtles. Between 1996 and 2016, we necropsied 127 apparently healthy pelagic olive ridley turtles (Lepidochelys olivacea) that died from drowning bycatch in fisheries and 44 live or freshly dead stranded turtles from the west coast of North and Central America and Hawaii. Seven percent (9/127) of pelagic and 47% (21/44) of stranded turtles had renal granulomas associated with S. Typhimurium. Stranded animals were 12 times more likely than pelagic animals to have Salmonella-induced nephritis suggesting that Salmonella may have been a contributing cause of stranding. S. Typhimurium was the only Salmonella serovar detected in L. olivacea, and phylogenetic analysis from whole genome sequencing showed that the isolates from L. olivacea formed a single clade distinct from other S. Typhimurium. Molecular clock analysis revealed that this novel clade may have originated as recently as a few decades ago. The phylogenetic lineage leading to this group is enriched for non-synonymous changes within the genomic area of Salmonella pathogenicity island 1 suggesting that these genes are important for host adaptation.

Pufferfish mortality associated with novel polar marine toxins in Hawaii.

Fish die-offs are important signals in tropical marine ecosystems. In 2010, a mass mortality of pufferfish in Hawaii (USA) was dominated by Arothron hispidus showing aberrant neurological behaviors. Using pathology, toxinology, and field surveys, we implicated a series of novel, polar, marine toxins as a likely cause of this mass mortality. Our findings are striking in that (1) a marine toxin was associated with a kill of a fish species that is itself toxic; (2) we provide a plausible mechanism to explain clinical signs of affected fish; and (3) this epizootic likely depleted puffer populations. Whilst our data are compelling, we did not synthesize the toxin de novo, and we were unable to categorically prove that the polar toxins caused mortality or that they were metabolites of an undefined parent compound. However, our approach does provide a template for marine fish kill investigations associated with marine toxins and inherent limitations of existing methods. Our study also highlights the need for more rapid and cost-effective tools to identify new marine toxins, particularly small, highly polar molecules.

MORTALITY PATTERNS IN ENDANGERED HAWAIIAN GEESE (NENE; BRANTA SANDVICENSIS).

Understanding causes of death can aid management and recovery of endangered bird populations. Toward those ends, we systematically examined 300 carcasses of endangered Hawaiian Geese (Nene; Branta sandvicensis) from Hawaii, Maui, Molokai, and Kauai between 1992 and 2013. The most common cause of death was emaciation, followed by trauma (vehicular strikes and predation), and infectious/inflammatory diseases of which toxoplasmosis (infection with Toxoplasma gondii) predominated. Toxicoses were less common and were dominated by lead poisoning or botulism. For captive birds, inflammatory conditions predominated, whereas emaciation, trauma, and inflammation were common in free-ranging birds. Mortality patterns were similar for males and females. Trauma predominated for adults, whereas emaciation was more common for goslings. Causes of death varied among islands, with trauma dominating on Molokai, emaciation and inflammation on Kauai, emaciation on Hawaii, and inflammation and trauma on Maui. Understanding habitat or genetic-related factors that predispose Nene (particularly goslings) to emaciation might reduce the impact of this finding. In addition, trauma and infection with T. gondii are human-related problems that may be attenuated if effectively managed (e.g., road signs, enforcement of speed limits, feral cat [Felis catus] control). Such management actions might serve to enhance recovery of this endangered species.

Molecular phylogeny of Babesia poelea from brown boobies (Sula leucogaster) from Johnston Atoll, central Pacific.

The phylogenetic relationship of avian Babesia with other piroplasms remains unclear, mainly because of a lack of objective criteria such as molecular phylogenetics. In this study, our objective was to sequence the entire 18S, ITS-1, 5.8S, and ITS-2 regions of the rRNA gene and partial beta-tubulin gene of B. poelea, first described from brown boobies (Sula leucogaster) from the central Pacific, and compare them to those of other piroplasms. Phylogenetic analyses of the entire 18S rRNA gene sequence revealed that B. poelea belonged to the clade of piroplasms previously detected in humans, domestic dogs, and wild ungulates in the western United States. The entire ITS-1, 5.8S, ITS-2, and partial beta-tubulin gene sequence shared conserved regions with previously described Babesia and Theileria species. The intron of the beta-tubulin gene was 45 bp. This is the first molecular characterization of an avian piroplasm.

Retrospective pathology survey of green turtles Chelonia mydas with fibropapillomatosis in the Hawaiian Islands, 1993--2003.

We necropsied 255 stranded green turtles Chelonia mydas with fibropapillomatosis (FP) from the Hawaiian Islands, North Pacific, from August 1993 through May 2003. Of these, 214 (84 %) were euthanized due to advanced FP and the remainder were found dead in fresh condition. Turtles were assigned a standardized tumor severity score ranging from 1 (lightly tumored) to 3 (heavily tumored). Tumors were counted and measured and categorized as external, oral, or internal and tissues evaluated by light microscopy. Turtles in tumor score 2 and 3 categories predominated, and tumor score 3 turtles were significantly larger than the other 2 categories. More juveniles stranded than subadults or adults. Total cross-sectional area of tumors increased significantly with straight carapace length (SCL). Frequency distribution of total number of external tumors per turtle was significantly skewed to the right, and there were significantly more tumors at the front than rear of turtles. Eighty percent of turtles had oral tumors, and 51% of turtles with oral tumors had tumors in the glottis. Thirty-nine percent of turtles had internal tumors, most of them in the lung, kidney and heart. Fibromas predominated in lung, kidney and musculoskeletal system whereas myxofibromas were more common in intestines and spleen. Fibrosarcomas of low-grade malignancy were most frequent in the heart, and heart tumors had a predilection for the right atrium. Turtles with FP had significant additional complications including inflammation with vascular flukes, bacterial infections, poor body condition, and necrosis of salt gland. Turtles with oral tumors were more likely to have secondary complications such as pneumonia. Most turtles came from the island of Oahu (74%) followed by Maui (20 %), Hawaii, Molokai, and Lanai (<3 % each). On Oahu, significantly more turtles we necropsied stranded along the northwestern and northeastern shores.

Protozoal and epitheliocystis-like infections in the introduced bluestripe snapper Lutjanus kasmira in Hawaii.

The bluestripe snapper, or taape, was introduced into Hawaii in the 1950s and has since become very abundant throughout the archipelago. As part of a health survey of reef fish in Hawaii, we necropsied 120 taape collected from various coastal areas south of Oahu and examined fish histology for extraintestinal organisms. Forty-seven percent of taape were infected with an apicomplexan protozoan compatible with a coccidian. Infection was evident mainly in the spleen and, less commonly, the kidney. Prevalence of this coccidian increased with size of fish, and we saw no significant pathology associated with the organism. Twenty-six percent of taape were also infected with an epitheliocystis-like organism that occurred mainly in the kidney and, less commonly, the spleen. In contrast to the coccidian, fish mounted a notable inflammatory response to the epitheliocystis-like organism, and this inflammation appeared to increase in severity with age. Prevalence of the epitheliocystis-like organism infection increased with age, but infection was not seen in fish greater than 26.5 cm fork length. The high prevalence of coccidial infection in introduced taape prompts the concern that these organisms, along with the epitheliocystis-like organism, have the potential to be transmitted to native reef fish. Given the impact of other introduced microbial organisms on native Hawaiian fauna, there is a clear need to assess whether protozoa and bacteria are endemic to Hawaii, and whether they negatively impact native reef fish that closely associate with taape.